Download Zebrafish as a Cancer Model

Subject Review
Zebrafish as a Cancer Model
Harma Feitsma and Edwin Cuppen
Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, the Netherlands
Abstract
The zebrafish has developed into an important model
organism for biomedical research over the last decades.
Although the main focus of zebrafish research has
traditionally been on developmental biology, keeping and
observing zebrafish in the lab led to the identification of
diseases similar to humans, such as cancer, which
subsequently became a subject for study. As a result,
about 50 articles have been published since 2000 in which
zebrafish were used as a cancer model. Strategies used
include carcinogenic treatments, transplantation of
mammalian cancer cells, forward genetic screens for
proliferation or genomic instability, reverse genetic
target-selected mutagenesis to inactivate known tumor
suppressor genes, and the generation of transgenics to
express human oncogenes. Zebrafish have been found to
develop almost any tumor type known from human, with
similar morphology and, according to gene expression
array studies, comparable signaling pathways. However,
tumor incidences are relatively low, albeit highly
comparable between different mutants, and tumors
develop late in life. In addition, tumor spectra are
sometimes different when compared with mice and
humans. Nevertheless, the zebrafish model has created
its own niche in cancer research, complementing existing
models with its specific experimental advantages
and characteristics. Examples of these are imaging
of tumor progression in living fish by fluorescence,
treatment with chemical compounds, and screening
possibilities not only for chemical modifiers but also
for genetic enhancers and suppressors. This review aims
to provide a comprehensive overview of the state
of the art of zebrafish as a model in cancer research.
(Mol Cancer Res 2008;6(5):685 – 94)
Cancer Research in Zebrafish
Zebrafish has been used as a laboratory animal for a few
decades now. Originally, the main focus was on developmental
biology because of the clear advantages of zebrafish such as
Received 11/20/07; revised 1/3/08; accepted 1/23/08.
Grant support: The Netherlands Cancer Genomics Center (Nationaal Regie
Orgaan Genomics).
Requests for reprints: Edwin Cuppen, Hubrecht Institute for Developmental
Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, the
Netherlands. Phone: 31-30-2121969; Fax: 31-30-2516554. E-mail: e.cuppen@
niob.knaw.nl
Copyright D 2008 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-07-2167
large clutch size, transparent embryos, and ex utero development of the embryo. While keeping zebrafish in the laboratory
environment, however, researchers observed different diseases
in adults, including cancer. Studies on the latter revealed that
zebrafish spontaneously develop almost any type of tumor
(1-4). The most common target tissues for spontaneous neoplasia are the testis, gut, thyroid, liver, peripheral nerve,
connective tissue, and ultimobranchial gland. Less common
target tissues include blood vessels, brain, gill, nasal epithelium,
and the lymphomyeloid system (2). In the first part of this
review, the currently used approaches to induce cancer in zebrafish will be discussed. An overview of these and their main
advantages and disadvantages are given in Table 1. Additionally, cancer results of the forward and reverse genetic and
transgenic mutant models are summarized in Table 2.
Treatment with Mutagens
Historically, researchers appreciated the relative ease of
treating fish with carcinogens because the chemicals can be
dissolved or suspended in water and the animals can be exposed
for longer time periods. When exposing zebrafish or guppies to
different compounds [e.g., 7,12-dimethylbenz(a )anthracene, N-nitrosodimethylamine, and N-nitrosodiethylamine],
mainly liver and intestinal tumors were observed (5-7). More
recently, similar but more extensive studies showed that
N-nitrosodiethylamine primarily induces liver and pancreas
carcinomas (8), and N-nitrosodimethylamine induces only liver
tumors (9). 7,12-Dimethylbenz[a]anthracene induces the broadest
tumor spectrum, including liver neoplasms; epithelial tumors in
intestine, pancreas, thyroid, and testis; mesenchymal tumors in
cartilage, blood vessels, muscles, and connective and lymphoid
tissues; and neural tumors (10). N-Methyl-N¶-nitro-N-nitrosoguanidine can also induce different tumor types, mainly liver
and testicular neoplasms, as well as hemangio(sarco)mas and
others (11). Some of these carcinogenic treatments have been
applied to mutants with a genetic predisposition to cancer but a
low spontaneous tumor incidence to show an increased sensitivity
of mutants compared with treated wild-type animals (12-14). Not
surprisingly, the alkylating mutagen N-ethyl-N-nitrosourea,
which introduces point mutations and is commonly used in
forward and reverse genetic screens, has also been found to induce
cancer. Following a group of animals from a mutagenesis screen,
Beckwith et al. (15) found that all fish developed skin papillomas
over time, but not invasive skin cancers.
Transplantation of Mammalian Cancer Cells into Zebrafish
Different groups have been experimenting with transplantation of mammalian cancer cells into zebrafish embryos. This
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Table 1. Comparison of Techniques to Induce Cancer in Zebrafish
Technique
Advantages
Disadvantages
Chemical treatment
Easy applicable
Large numbers of fish
Long-term treatment
Rapid onset
Study in transparent embryos
Use of human cancers
Use of fluorescence
Identification of multiple genes at once
Embryonic phenotype as readout
Combination with drugs, mutants or transgenics
Inactivation of one specific gene
Human-like cancer mutations
Unspecific
Predominance of liver tumors
Potential hazard for researcher
Cannot be propagated as a line
Low penetrance of tumors
Transplantation of mammalian cells
Forward genetic screens
Reverse genetic knockouts
Expression of transgenes
Laborious
Screens for adult phenotypes require space and time
Stochasticity of cancer makes it difficult as readout
Background mutations
Late onset and low penetrance
Gene duplications
Different tumor spectrum than in humans
Laborious if no transgenic line can be established
Lack of specific promoters
Easy generation by injection
Rapid onset
Use of human genes
Use of fluorescence
Conditional and tissue-specific expression
creates an in vivo system in which the advantages of cultured
human cancer cells are combined with those of transparent
zebrafish embryos in which development can be followed. Lee
et al. (16) transplanted fluorescently labeled human metastatic
melanoma cells into zebrafish blastula-stage embryos and
showed that these cells survive, migrate, and divide, and are
still present in adults but do not cause cancer or metastases.
Another study showed that aggressive human melanoma cells
are able to induce a secondary axis or an abnormal head when
transplanted into 3-hour-old zebrafish embryos, which was
shown to be due to Nodal signaling from the tumor cells
(17, 18). In contrast to the above studies where no cancer
development was observed, similar human melanoma cells as
well as a colorectal and a pancreatic cancer cell line were found
to induce tumor-like cell masses when transplanted into 2-dayold zebrafish embryos (19).
Transplantation studies can be specifically effective in the
study of vasculature remodeling, cancer invasion, and metastasis. Currently, no good in vivo model is available in which
such dynamic process can be followed in real time. When
transplanted into 2-day-old zebrafish embryos, human and
murine tumor cell lines expressing fibroblast growth factor or
vascular endothelial growth factor induced rapid neovascularization inside the tumor graft, and this could be inhibited by
treatment with antiangiogenic chemicals (20). In another elegant
study, researchers used i.p. injection of fluorescently labeled
human breast cancer cells in 1-month-old zebrafish in combination with three-dimensional modeling to show how the human
cells interact with vessels and invade in tissues (Fig. 1C). They
showed that expression of vascular endothelial growth factor
induces openings in vessel walls that can be used for invasion,
which in turn is stimulated by RhoC expression (21).
Table 2. Cancer Mutants in Zebrafish
Model
Cancer
Forward genetic mutants
Ribosomal protein
separase
bmyb
Genomic instability
Reverse genetic mutants
tp53
ptenb
apc
mlh1, msh2, msh6
Transgenic mutants
RUNX1-CBF2T1
TEL-AML1
Zebrafish tel-jak2
Mouse c-myc
Mouse c-myc (line)
Mouse c-myc (conditional)
Zebrafish bcl2
NOTCH
kRASG12D
kRASG12D (conditional)
BRAF-V600E
MYCN
Type
Incidence (%)
Onset (mo)
Ref.
+
Induced
Induced
+
MPNST
Liver, intestinal
Testis, vascular
MPNST and others
35
10
7
Up to 48
8-24
3-12
3-12
30-36
(24)
(13)
(14)
(25)
+
+
+
+
MPNST
Ocular
Liver, intestinal
MPNST and others
28
33
29
33
14
7-18
15
17
(27)
(28)
(12)
+
Leukemia
3
8-12
+
+
+
Leukemia
Leukemia
Leukemia
6
100
81
1-4
2
4
Leukemia
Rhabdomyosarcoma
Rhabdomyosarcoma and others
Melanoma
Neuroendocrine
40
47
100% of survivors
6
2
5-11
2
1
4
5
+
+
+
In tp53 /
+
(32)
(33)
(34)
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
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Zebrafish as a Cancer Model
FIGURE 1. The unique possibilities of zebrafish can be used to study different stages of cancer development. A. Genome instability will lead to mutation
accumulation that can result in cancer. Here, somatic loss of heterozygosity of the albino gene is shown in a patch of cells (arrow ) in the pigment layer of the
eye of an msh6 mutant zebrafish embryo (H. Feitsma, A. Akay, E. Cuppen, unpublished results). B. Cancer growth can be followed in living adult zebrafish.
Here, the growth of a green fluorescent protein – labeled T-cell acute lymphoblastic leukemia in stable transgenic fish is shown. Top, rag2-EGFP control fish;
bottom, rag2-EGFP-mMyc fish with massive leukemia; T, thymus. [Reprinted with permission from ref. 36; copyright (2005) National Academy of Sciences.]
C. Invasion of cancer cells in blood vessels is the first step of metastasis. Here, transplanted dsRED-labeled human cancer cells penetrate green fluorescent
protein – labeled blood vessels (arrows ) in a zebrafish. [Reprinted with permission from ref. 21; copyright (2007) National Academy of Sciences.].
Mutants from Forward Genetic Screens
The largest impetus for zebrafish to become an important
animal model was its suitability for forward genetic screens.
Since the first mutagenesis experiments in the Nusslein-Volhard
lab (22), screens have been carried out for almost any type of
phenotype, including phenotypes related to cancer (Table 2).
Amsterdam et al. (23) rather coincidentally ran into a set
of genes that cause cancer when mutated. Using retroviral
insertions, they carried out a screen for embryonic lethality, but
while keeping the heterozygous founders they noticed that 12
of their lines displayed an elevated tumor incidence (24).
Eleven of those lines carried insertions in ribosomal genes, for
which there was no strong relation with cancer at that point.
The other line was mutated in one of the two zebrafish
homologues of the neurofibromatosis 2 (nf2) gene, a known
tumor suppressor in humans.
The Zon lab conducted a screen for proliferation defects,
taking advantage of the fact that many oncogenes and tumor
suppressors are actually essential for development. Homozygous mutant embryos are therefore lethal, and heterozygotes
are, due to haploinsufficiency or loss of heterozygosity,
predisposed to cancer later in life. Two mutants from this
screen have been reported thus far, a loss-of-function mutation
in bmyb (14) and a loss-of-function mutation in separase (13),
genes that both had not previously been unequivocally
implicated in cancer. Homozygous mutant embryos of both
displayed mitotic defects and genomic instability, whereas
heterozygous adults showed a marginal increase (2- to 2.5-fold)
in cancer incidence when treated with N-methyl-N¶-nitroN-nitrosoguanidine.
An elegant screen for genomic instability used loss of
heterozygosity of the golden pigmentation gene as a readout
(25). When in golden heterozygous embryos, cells of the retinal
pigment epithelium of the eye acquire a mutation in the wildtype allele. This results in unpigmented patches that are easily
scorable in large batches of embryos (Fig. 1A). The 12 mutant
lines with embryonic genomic instability that were obtained all
showed some or more sensitivity to cancer in heterozygous
adults, confirming the strong connection between genomic
instability and cancer. However, the underlying mutations have
not been mapped or cloned yet.
Reverse Genetics: Target-Selected Inactivation of Tumor
Suppressor Genes
Since knockout technology in zebrafish became available by
means of target-selected mutagenesis (26), several mutants for
known tumor suppressor genes have been generated (Table 2).
For tp53, the most frequently mutated gene in human cancers,
two zebrafish mutants were isolated: one with a missense
mutation in the DNA-binding domain (tp53M214K) and one with
a missense mutation that affects protein structure in a heatsensitive manner (tp53N168K). Homozygotes of both lines were
developmentally normal but showed suppressed apoptosis on
irradiation. Twenty-eight percent of tp53M214K homozygotes
developed tumors at an average age of 14 months, which,
except for one melanoma, were all diagnosed as malignant
peripheral nerve sheath tumors (MPNST; ref. 27).
The second most frequently mutated tumor suppressor in
human cancers, pten, has undergone a gene duplication in
zebrafish. Faucherre et al. isolated nonsense mutants for both
ptena and ptenb. Single homozygous mutants of either allele
did not display a developmental phenotype, but mutants lacking
both ptena and ptenb died at day 5 postfertilization from
pleiotrophic defects due to enhanced proliferation and cell
survival, indicating redundant functions. However, adult fish
lacking only ptenb developed ocular tumors with an incidence
of 33% at 18 months, whereas no neoplasms were observed in
adult ptena mutants. Tumors were not further classified, but
their appearance and the fact that they occur only intraocular
suggest that they are not MPNSTs (28).
Mutations in the adenomatous polyposis coli (APC) gene
cause the vast majority of human sporadic and inherited
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colorectal cancers by constitutively activating the Wnt signaling
pathway (29). A nonsense mutation in zebrafish apc was found
to result in lethality when homozygous (30). Less than 30% of
heterozygous fish spontaneously developed liver and intestinal
tumors from 15 months of age onward. Treatment of apc
heterozygotes with 7,12-dimethylbenz[a]anthracene enhanced
tumorigenesis, resulting in intestinal, hepatic, and pancreatic
tumors, with frequencies 3- to 4-fold higher than treated wildtypes. The tumors displayed activated Wnt signaling, indicating
that the genetic pathway is conserved (12).
The first DNA repair genes that have been mutated in
zebrafish are the mismatch repair genes mlh1, msh2, and msh6,
which are involved in the repair of small replication errors such
as base mismatches and insertion/deletion loops. Homozygous
mutants were genomic unstable, as shown by the occurrence of
variation in lengths of microsatellite sequences in their DNA.
Taking the data for the three mutants together, 33% of
homozygotes, on average, developed tumors at an average
age of 17 months. Mainly MPNSTs were found at different
places in the body, but other tumor types were also observed.1
Targeted knockout strategies such as those used for making
mouse knockouts are not available in zebrafish. The current
strategy makes use of random N-ethyl-N-nitrosourea mutagenesis combined with targeted selection of mutations in the gene
of interest, which means that the researcher is dependent on
the random point mutations that are induced. However, the
positive aspect of this is that the generated point mutations
can be more similar to the type of spontaneous mutations that
occur in human cancer patients than the large gene deletions or
insertions in mouse knockouts. Indeed, two of the published
zebrafish mutants do exactly mimic human cancer mutations:
the tp53M214K point mutation in zebrafish tp53 is frequently
found in human cancers (27) and the splice site mutation in the
zebrafish msh2 gene results in an in-frame loss of exon 5,1
which is one of the most frequent familial mutations in
hereditary nonpolyposis colorectal cancer, the human syndrome
that is caused by defective mismatch repair.
One of the disadvantages of the use of random mutagenesis
in the knockout procedure is that each fish that is retrieved will
contain, besides the mutation of interest, several background
mutations. Those additional mutations are heterozygous and will
probably not have a large effect on developmental phenotypes,
but they may of course be of influence on the process of
mutation accumulation that is necessary for tumor development.
Therefore, N-ethyl-N-nitrosourea – induced mutants should be
crossed out for several generations to exclude confounding
effects of unknown background mutations.
Transgenic Zebrafish Expressing Mammalian Oncogenes
The largest number of studies on cancer development in
zebrafish thus far come from transgenic zebrafish expressing
mammalian oncogenes (Table 2). This approach makes use of
another advantage of zebrafish as a laboratory animal: the
convenience of introducing foreign DNA into zebrafish cells
and getting it expressed by injection into one-cell embryos.
1
H. Feitsma, R.V. Kuiper, J. Korving, I.J. Nijman, E. Cuppen, submitted for
publication.
Many of the models concern lymphomas and leukemias,
cancers that rarely occur spontaneously in zebrafish but for
which the transgenic model may be of great aid in searching for
new treatments. This is especially important for this class of
diseases when considering that human patients usually need
therapies severely affecting quality of life.
Frequently, hematologic malignancies arise because of
genetic rearrangements resulting in misexpression of certain
oncogenes (e.g., by fusion to lymphoid-expressed genes).
Although many of the genes and even chromosome regions
known to be involved have been conserved in zebrafish (31),
researchers have mostly limited their experiments to expressing
human fusion constructs. For example, injection of a human
RUNX1-CBF2T1 fusion, which is frequent in acute myeloid
leukemia, caused circulation defects, hemorrhages, abnormal
vascular development, and defective hematopoiesis, but not
leukemia, in zebrafish embryos (32). The human TEL-AML1
fusion is responsible for 25% of childhood pre-B acute
lymphoblastic leukemia cases. When expressing a TELAML1 fusion construct in zebrafish, acute lymphoblastic
leukemia developed in 3% of fish that expressed the transgene
ubiquitously, but not when expression was restricted to
lymphoid cells using the rag2 promotor (33). In an attempt
to model a molecular rearrangement associated with human
lymphoblastic and myeloid leukemias, Onnebo et al. (34)
injected embryos with a zebrafish tel-jak2 fusion under control
of the myeloid spi1 promotor, which caused not only severe
perturbation of hematopoiesis but also high mortality, making it
impossible to study adults.
Langenau et al. have put an enormous effort into generating
optimal zebrafish models for leukemia. They developed
zebrafish expressing mouse c-myc under the zebrafish rag2
promotor to restrict the expression to lymphoid cells. MYC is
known to play an important role in human and mouse lymphoid
malignancies. Six percent of injected fish developed infiltrative
T-cell leukemia (Fig. 1B) with a latency of 30 to 131 days (35).
A germ line – transmitting transgenic line of these fish displayed
100% cancer incidence with a mean latency of 80 days (36), but
because disease onset mostly precedes reproductive age, the
line can only be propagated via in vitro fertilization, which
makes experiments with this model labor intensive. However,
by creating a conditional transgene in which a dsRED gene
flanked by loxP sites was put in between the rag2 promotor and
the mouse c-myc (mMyc) gene (rag2-loxP-dsRED-loxP-EGFPmMyc), and using this transgenic line in combination with CRE
injection, leukemia development was made inducible. Unfortunately, only partial recombination was obtained, resulting in
leukemia in only 12 of 186 CRE-injected animals (36). A
second improvement was made by crossing the conditional line
to a transgenic line expressing CRE under a heat shock promotor (37). On heat shock, double transgenic fish developed
T-lymphoblastic lymphoma that rapidly progressed to T-cell
acute lymphoblastic leukemia. The best heat shock treatment
gave an induction of cancer in 81% of fish, with a mean latency
of 120 days, but in the next generations of the transgenic line
the cancer latency was higher (37). The same lab also developed a transgenic line overexpressing the zebrafish B-cell
leukemia 2 (bcl2) gene under the rag2 promotor, in which
lymphoid apoptosis is blocked (38). Irradiation treatment of fish
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Zebrafish as a Cancer Model
that were transplanted with leukemic cells from mMyc
transgenic fish (36) ablated cancer in wild-type fish but not in
the bcl2-overexpressing fish (38). In another leukemia model,
the knowledge was used that activating mutations in NOTCH1
are found in f60% of T-cell acute lymphoblastic leukemia.
Zebrafish expressing constitutively active human NOTCH
under the rag2 promotor developed T-cell leukemia at 5 to
11 months in f40% of the cases (39). When crossing these fish
with the bcl2-overexpressing line (38), disease onset was
accelerated and the leukemias were not radiation sensitive
anymore, as apoptosis was inhibited.
Several transgenic models for solid tumors have also been
generated. Langenau et al. observed that the zebrafish rag2
promotor that they frequently used showed ectopic expression in
undifferentiated cells of the muscle, making it possible to express
human activated RAS (kRASG12D), a common mutation in
human oncogenesis, in these cells, resulting in the development
of rhabdomyosarcoma in 47% of the cases by 80 days of age and
in only one lymphoid hyperplasia (40). Tumorigenesis was
accelerated when these experiments were done on the tp53
mutant genetic background. A conditional version of the same
activated human RAS under a zebrafish b-actin promoter with
a floxed EGFP gene inserted in between was combined with
the previously mentioned heat shock– inducible CRE. This
resulted in overall reduced viability in heat-shocked as well as
non – heat-shocked animals. The latter is most likely due to
leakiness of the heat shock promotor of the CRE line. Surviving
fish were found to develop four types of tumors at f35 days:
rhabdomyosarcomas, myeloproliferative disorders, intestinal
epithelial hyperplasms, and two cases of MPNST (41).
To test the role of activated BRAF in melanoma development,
transgenic zebrafish expressing activated human BRAF-V600E
under the zebrafish melanocyte mitfa promotor were generated.
These fish developed pigmented nevi in 10% of the cases, but not
melanoma (42). Although tp53 mutations are rare in human
melanoma, in tp53 mutant background 4 of 66 transgenic fish
developed highly invasive and transplantable melanoma.
Finally, human MYCN under the zebrafish myod promotor
is expressed in neural tissue and pancreas and, in combination
with a human core enhancer, also in muscles. Only 5 of
250 fish injected with this construct developed tumors at f5
months of age, one cranial and four abdominal, but all
neuroendocrine (43).
Zebrafish Cancers Compared with Mammalian
Cancers
As previously mentioned, zebrafish can develop almost any
type of cancer (2). Moreover, a tremendous asset of zebrafish as
a cancer model is that many tumors histologically resemble
human tumors (Fig. 2; ref. 44). In addition, more general cancer
characteristics such as genomic instability, invasiveness,
transplantability, and the existence of cancer stem cells (40)
apply to zebrafish tumors as well (Fig. 1), and many tumor
suppressor genes and oncogenes have been conserved. Taken
together, these studies validate zebrafish as a bona fide cancer
model. However, many details are still unknown and some
important differences with regard to human tumorigenesis have
also become clear, which will be discussed below.
Cancer Incidence and Onset
The tumor suppressor genes that have been mutated in
zebrafish have thus far been limited to the few well-studied
classic examples, for which mouse knockouts have been
generated as well, and human hereditary diseases are known.
Mouse apc(D716) knockouts are embryonic lethal in the
homozygous state, similar to what was found in zebrafish.
However, all heterozygous mice develop intestinal polyps by
7 weeks of age (45). Pten homozygous mutant mice are also
early embryonic lethal, and heterozygotes die before 3.5 months
of age from malignant tumors (46). Likewise, 74% of p53
homozygous knockout mice develop cancer before 6 months of
age (47). The msh2 and mlh1 mouse knockouts were found to
have a 50% cancer incidence at 6 months. For the msh6
knockout this was less severe because msh6 is partially
redundant with msh3 (48). The conditions caused by mutations
in the above genes in humans are Li-Fraumeni syndrome for
TP53 (49), familial adenomatous polyposis for APC (50),
Cowden disease and Bannayan-Riley-Ruvalcaba syndrome for
PTEN (51), and hereditary nonpolyposis colorectal cancer for
mismatch repair genes (52). These human diseases are considered early-onset syndromes, becoming clinically relevant
around the age of 40 to 45 years. Tumor incidences are probably
high but mostly unknown because unaffected carriers of disease
mutations are usually not noticed.
Overall, the tumor incidences in zebrafish are generally
lower and the onset is later as compared with the orthologous
mouse models. Although animal numbers of the initial studies
are limited, it turns out that the cancer incidence is quite similar
for all zebrafish mutants, close to 30% (12, 27, 28).1 In
addition, the average tumor frequency in the ribosomal gene
mutant lines of the retroviral insertion screen was also f35%
(24). This high similarity in frequencies is remarkable, and
suggests that this is the maximum frequency of cancer that
spontaneously develops in genetically predisposed zebrafish.
Furthermore, zebrafish develop tumors generally in the second
year of life (12, 27, 28),1 whereas the comparable mouse
mutants develop cancer within the first 6 months of life.
Because the overall life span is similar for mice and fish, the
difference could be due to the lower number of cells in fish,
which simply decreases the chance that one body cell will
acquire all oncogene activations and tumor suppressor truncations necessary for tumor development. However, considering the zebrafish body weight is f100-fold lower than that of
mouse (0.3 versus 30 g) and the number of cells will be more or
less accordingly, a time to disease onset that is four times larger
(2 years versus 6 months) and a disease incidence that is three
times smaller (30% versus 100%) are actually relatively small
differences. In addition, the lower zebrafish disease incidence
as compared with mice is experimentally not a major problem
because generating and keeping a 3-fold larger number of fish is
practically easy. In any case, the frequency of cancer in mutant
fish is much higher than in wild types, which spontaneously
develop tumors in the order of 0% (27) to 11% (24) of the
population. Another long-term study of spontaneous neoplasms
determined that wild-type fish have 1% cancer incidence in
the first year, which increases in the second year (10). However,
most groups agree that more data need to be generated on tumor
incidence in wild-type fish to improve the model.
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FIGURE 2. Zebrafish tumors resemble various types of human tumors at the histologic level. Comparison of histology of neurofibroma (left ; stained
with H&E), squamous cell carcinoma (middle; stained with H&E), and intestinal adenoma (right ; immunostained for b-catenin ) in zebrafish (top ) and
human (bottom ).
Carcinogen treatments on zebrafish give more robust induction of cancer and are considerably easier to perform as compared
with the mouse system. Although early studies of transgenic fish
expressing oncogenes yielded highly variable tumor incidences,
it is exciting and promising to see that technical improvements
boosted cancer incidences to 80% to 100% (36).
A recurrent problem in zebrafish is the presence of
duplicated genes resulting from a recent partial or complete
genome duplication in teleosts (53), which may influence the
role of oncogenes and tumor suppressors in carcinogenesis.
An example is the presence of two forms of pten in the
zebrafish genome, which were found to be functionally
redundant in development but not in oncogenesis: no loss of
ptena was observed in tumors of ptenb homozygous mutants
(28). In addition, although loss of heterozygosity is expected
to be the frequent mechanism in humans to explain the cancer
predisposition in individuals with heterozygous tumor suppressor mutations (54), it was hardly ever observed in the
comparable zebrafish mutants: not in apc (12), not in all
mutant lines of the retroviral insertion screen (24), only once
in the whole set of genomic instability mutants (25), twice in
the separase mutant (interestingly, these particular mutants did
not show a polyploidy phenotype; ref. 13), and never in the
bmyb mutant (14). An interesting study in this respect is the
N-nitrosodimethylamine induction of liver tumors in triploid
and diploid zebrafish (9). When assuming that loss of function
of tumor suppressor genes is an important step in cancer development, one would expect that in case of a triploid fish,
three alleles need to be hit to lose the function of the gene,
consequently taking longer for tumors to develop. Indeed,
triploid fish had a slightly later onset of cancer, but
concomitantly these fish showed a higher incidence of tumors,
which indicates that activation of oncogenes is equally
important.
Tumor Spectrum
Most tumor classifications in zebrafish are still rather broad,
lacking the more sophisticated identification marker stainings
available for mammals. Unfortunately, many commonly used
assays for human and mouse do not work on zebrafish material,
despite repeated efforts from probably almost many zebrafish
tumorigenesis labs. The Cheng lab is working towards a
systematic zebrafish tumor histology database,2 which is a very
valuable initiative to standardize tumor classification. Nevertheless, the need for zebrafish-specific antibodies is evident for
this and many other research areas.
The most common target tissues for spontaneous tumors in
wild-type fish are testis and liver (2). In contrast, genetic mutant
lines most commonly develop MPNSTs, as observed in the p53,
mismatch repair, ribosomal protein, and genomic instability
mutants (24, 25, 27).1 Likewise, the transgenic line overexpressing human MYCN develops MPNSTs (43). MPNSTs
form a large group of tumors that includes, for example,
neurofibromas, the tumor type that occurs in human neurofibromatosis conditions (Fig. 2). In the nf2 mutant fish line from the
retroviral screen, this type of tumors can therefore certainly be
expected (24). As already mentioned, defective mismatch repair
in humans is linked to hereditary nonpolyposis colorectal cancer,
which is a colorectal cancer. However, this concerns patients
that are heterozygous for the mismatch repair gene mutation.
Interestingly, the rare patients with biallelic inactivation of
mismatch repair genes have been reported to develop a more
neurofibromatosis type 1 – like syndrome with skin neurofibromas and brain tumors. Mouse mismatch repair knockouts
mainly develop lymphomas (48), indicating that the zebrafish
models important aspects of the human disease that are not
2
K. Cheng, personal communication.
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Zebrafish as a Cancer Model
observed in mice (Table 3). p53 mutants in human and mouse do
not develop MPNSTs but rather osteosarcomas, rhabdomyosarcomas, breast cancer, brain tumors, and leukemias in human
(49), and lymphomas and sarcomas in mouse (Table 3; ref. 47).
The intestinal and liver tumors in zebrafish apc mutants (12)
are comparable to those in mouse mutants and human patients,
also with regard to constitutive activation of the Wnt pathway in
tumors (Table 3; Fig. 2; refs. 45, 50). Because the zebrafish gut is
much smaller than that of mouse and human, the absolute chances
for developing gut cancer are probably proportionally smaller,
possibly explaining the observed difference in penetrance.
Surprisingly, in zebrafish ptenb mutants, only one type of tumors
inside the eye was found (28), whereas mouse and human pten
mutants show a broad spectrum of cancers (Table 3; refs. 46, 51).
To get a more detailed view on similarities of fish and
human cancers, gene expression profiles of zebrafish and
human tumors have been compared. For the bmyb mutant, gene
expression profiles of homozygous embryos showed significant
correlation with human cancers (14), which is noteworthy, as a
developmental zebrafish phenotype was compared with a
human cancer. A more extensive expression array study was
done on 7,12-dimethylbenz[a]anthracene – and 1,4-diphenyl1,3-butadiene – induced liver tumors in zebrafish, showing
significant similarities with human liver tumors, but not with
other human tumor types (55-57). The two human types of
rhabdomyosarcoma, embryonic rhabdomyosarcoma and alveolar rhabdomyosarcoma, have a distinct molecular signature.
Microarray analysis on rhabdomyosarcomas of activated RAS
transgenic fish showed that these have a signature similar to
human embryonic rhabdomyosarcoma but not to alveolar rhabdomyosarcoma. The profile also showed similarities to other
RAS-induced tumors such as human pancreatic adenocarcinoma and mouse lung adenocarcinoma, in addition to a more
specific embryonic rhabdomyosarcoma signature of muscle
genes (40). These studies collectively indicate that the genetic
pathways involved in cancer development are conserved
between fish and mammals.
Specific Advantages of Zebrafish
The differences of zebrafish cancer from mammalian cancers
do not compromise the organism as a cancer model, but should
rather help focusing on the specific strengths of zebrafish to unravel
mechanisms in carcinogenesis, complementary to other models.
Screens
The suitability of zebrafish for genetic screens has long been
recognized also in cancer research. However, although very
elegant strategies were chosen for the first screens for cancer
genes that were done in zebrafish, the results were promising
but not completely convincing. Two new genes for which a
relation to cancer was unknown or unclear were identified in
the proliferation screen, but the cancer predisposition in
heterozygous mutants is marginal and only visible when
carcinogen treatment was used (13, 14). In relation to this, no
mutations in separase, one of the identified genes, were found
in 82 human tumor cell lines. In addition, although the
ribosomal protein mutants from the retroviral insertion screen
were clearly cancer-prone, a clear connection of this important
and large group of genes to cancer had never been observed in
other organisms (24). Certainly, further research is necessary to
qualify these genes as real ‘‘cancer genes.’’ The mutants from
the genomic instability were all clearly predisposed to cancer
(25), but because the embryonic phenotype is a matter of
chance and not fully penetrant, mapping the mutants is labor
intensive and time-consuming. Nevertheless, there are many
options for advanced novel screens by performing mutagenesis
in a cancer-prone background or crossing mutants to a cancerprone line to identify genetic modifiers of specific cancers.
Additionally, making use of transgenic lines with fluorescent
reporters and/or chemical compound libraries (see below) will
certainly result in new steps in cancer research.
Imaging
The lauded advantage of zebrafish embryos being transparent
does not, except for some transplantation studies, apply to most
cancer studies in zebrafish that involve adult animals. However,
a relatively transparent adult zebrafish line that lacks all types of
pigment has been generated by Znomics (58). Additionally,
Goessling et al. (59) have successfully applied high-resolution
microscopic ultrasound to follow tumor development and
regression by treatment in living adult fish. Other existing
imaging techniques that should, in principle, be possible in
zebrafish are tomography and magnetic resonance imaging (58).
Most effective, however, and specific for zebrafish is the use of
fluorescence, as even adult zebrafish are small enough to be able
to visualize fluorescent organs or tumors inside the living body.
Tumor development and cancer spreading can therefore be
followed over time in a living fish. Indeed, transplantation studies
described above often used fluorescently labeled mammalian
cells, facilitating monitoring of behavior inside the host (16,
17, 19). Furthermore, by combining transgenic zebrafish lines
expressing fluorophores in the vasculature with transplantation of
differentially labeled mammalian cancer cells, the dynamics
of vascular remodeling within the tumor can be visualized in a
detailed fashion over time in a living model (20, 21), which is
a unique opportunity for this research field (Fig. 1C).
Table 3. Cancer Types in Tumor Suppressor Mutants in Zebrafish, Mouse, and Human
Gene
Zebrafish
Mouse
Human
TP53
PTEN
APC
MMR
MPNSTs
Intraocular tumors
Liver and intestinal tumors
MPNSTs and others
Lymphomas and sarcomas
Broad spectrum
Intestinal polyps
Lymphomas and gastrointestinal tumors
Sarcomas, breast cancers, brain tumors, leukemias (Li-Fraumeni)
Broad spectrum
Colorectal cancer (FAP)
Heterozygous: colorectal cancer (HNPCC)
Homozygous: brain tumors and neurofibromas, lymphomas and leukemias
Abbreviations: FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal cancer.
Mol Cancer Res 2008;6(5). May 2008
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691
692 Feitsma and Cuppen
Another example of the versatility of fluorescence in zebrafish is the generation of transgenic zebrafish lines expressing
mammalian oncogenes, where the expression cassettes are
usually provided with fluorescence markers to visualize the
carcinogenesis process (Fig. 1B; refs. 33, 35, 39, 41, 43). For
example, Langenau et al. (38) used fluorescence of labeled
leukemic cells to show that radiation treatment of wild-type fish
transplanted with leukemia cells resulted in disappearance of
the cancer, whereas in bcl2-overexpressing, apoptosis-deficient
fish the cancer remained present. Fluorescence was also very
elegantly used for tracking recombination events in cre/loxregulated systems in whole fish. A loxed dsRED gene in
between the rag2 promotor and EGFP-mMYC transgene
resulted in dsRED expression in thymocytes, but on recombination in the presence of CRE, resulted in a switch to enhanced
green fluorescent protein (EGFP) expression in the same cells
(36, 37). In the rhabdomyosarcoma model mentioned above,
different fluorescent markers were successfully used for the
fluorescence-activated cell sorting of four distinguishable cell
types to identify cancer stem cells that have the capacity to
induce new cancers in transplanted fish (40).
The above examples are pioneering studies, but they already
show the enormous possibilities of using fluorescence in
zebrafish cancer research, being indicative of a proportional
number of opportunities for new studies. For example, some of
the transgenic lines will be very well suited for genetic or
chemical screens to identify modifiers of carcinogenesis,
potentially in automated high-content screening setups.
Chemical Treatments in Search for Drugs
As already mentioned, chemical treatment of both embryos
and adult animals is relatively easy for zebrafish. In this
respect, zebrafish can be a versatile model in the search for
cancer therapeutics. Some proof of principle comes from the
effective use of known angiogenesis inhibitors in transplantation-induced vascular remodeling. The neovascularization
inside mammalian tumor grafts in zebrafish embryos could
be inhibited by treatment with antiangiogenic drugs, whereas
development of the normal vasculature in these embryos was
not influenced (20). One of those compounds was also shown
to block the angiogenic response to human tumor cell –
secreted vascular endothelial growth factor in zebrafish
embryos (21). Another type of search for therapeutics concerns
that of radioprotective agents, which are of great clinical
relevance considering the importance of radiotherapy in human
cancer treatment. As an example, the nanoparticle DF-1 was
shown to reduce ionizing radiation damage in zebrafish
embryos (60).
The state of the art would be to screen chemical compound
libraries to identify novel drugs that inhibit certain aspects of
cancer development. For this purpose, mutants that are
embryonic lethal in the homozygous state and cancer-prone in
the heterozygous state, such as those from the retroviral
insertion and proliferation screens or the apc mutant (12-14,
24, 30), can be very useful because the early embryonic phenotype can be used as a readout whereas the obtained
compound may very well be applicable to the adult cancer
phenotype. In a nice and successful example of this, the bmyb
mutant was used for a small-molecule screen in which the novel
compound persynthamide was found to rescue its embryonic
phenotype (61, 62). Unfortunately, the effectiveness of this
compound in adults has not yet been reported. A similarly nice
small-molecule screen identified the compound 4-bromo3-nitropropiophenone as a radiation sensitizer specifically for
cancer cells. In zebrafish embryos transplanted with human
cancer cells and treated with this compound, tumor growth was
inhibited by irradiation while there was no effect on embryonic
development (63).
Outlook
Although the area of cancer research in zebrafish is still
relatively young, from this overview, it becomes clear that
enormous progress has been made since the year 2000.
Versatile tools and infrastructure for studying various aspects
of carcinogenesis have become available and have been
validated to various degrees. However, new possibilities may
be contained in technologies that have successfully been used
in cancer research in other models but are not yet established
for zebrafish. For example in transgenesis, elegant systems for
spatial and temporal control of gene expression, such as those
driven by tetracycline or using GAL4 upstream activating
sequences, are available in fruitfly and mouse (64, 65). Tetracycline-responsive systems (Tet-On and Tet-Off) allow for on
and off switching of gene expression, which, when combined
with oncogenes or tumor supressors, would give the opportunity to follow tumor progression and regression in zebrafish.
The GAL4 upstream activating sequence system makes use of
endogenous enhancers to target expression. Because the
currently available tissue-specific promotors in zebrafish are
limited and sometimes unspecific, generating a collection of
specific enhancer-trap lines, such as successfully done by the
Becker lab (66), would be valuable in strategies to target cancer
gene expression. Nevertheless, whereas more universal standards are being developed, many opportunities are now on hand
for exciting cancer-related studies in this small but multipurpose vertebrate model. As a result, the zebrafish can be
expected to contribute to novel insights in tumor biology and
cancer drug development in the near future.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Rachel Giles and Jeroen den Hertog for helpful comments on the
manuscript; David Langenau, Richard Klemke, Wim Spliet, Anna Haramis, and
Johan van Es for kindly supplying photographs; and Keith Cheng for personal
communication about the zebrafish cancer database effort.
References
1. Kent ML, Bishop-Stewart JK, Matthews JL, Spitsbergen JM. Pseudocapillaria
tomentosa, a nematode pathogen, and associated neoplasms of zebrafish (Danio
rerio ) kept in research colonies. Comp Med 2002;52:354 – 8.
2. Kent ML, Spitsbergen JM, Matthews JM, Fournie JW, Westerfield M. Diseases
of zebrafish in research facilities. Zebrafish International Resource Center. 2002
Sept. Available from: http://zebrafish.org/zirc/health/diseaseManual.php.
3. Matthews JL. Common diseases of laboratory zebrafish. Methods Cell Biol
2004;77:617 – 43.
4. Smolowitz R, Hanley J, Richmond H. A three-year retrospective study of
abdominal tumors in zebrafish maintained in an aquatic laboratory animal facility.
Biol Bull 2002;203:265 – 6.
Mol Cancer Res 2008;6(5). May 2008
Downloaded from mcr.aacrjournals.org on May 10, 2017. © 2008 American Association for Cancer Research.
Zebrafish as a Cancer Model
5. Khudoley VV. Use of aquarium fish, Danio rerio and Poecilia reticulata ,
as test species for evaluation of nitrosamine carcinogenicity. Natl Cancer Inst
Monogr 1984;65:65 – 70.
6. Pliss GB, Khudoley VV. Tumor induction by carcinogenic agents in aquarium
fish. J Natl Cancer Inst 1975;55:129 – 36.
7. Stanton MF. Diethylnitrosamine-induced hepatic degeneration and neoplasia in the aquarium fish, Brachydanio rerio . J Natl Cancer Inst 1965;34:
117 – 30.
8. Mizgireuv IV, Revskoy SY. Transplantable tumor lines generated in clonal
zebrafish. Cancer Res 2006;66:3120 – 5.
9. Mizgireuv IV, Majorova IG, Gorodinskaya VM, Khudoley VV, Revskoy SY.
Carcinogenic effect of N-nitrosodimethylamine on diploid and triploid zebrafish
(Danio rerio ). Toxicol Pathol 2004;32:514 – 8.
10. Spitsbergen JM, Tsai HW, Reddy A, et al. Neoplasia in zebrafish (Danio
rerio) treated with 7,12-dimethylbenz[a ]anthracene by two exposure routes at
different developmental stages. Toxicol Pathol 2000;28:705 – 15.
11. Spitsbergen JM, Tsai HW, Reddy A, et al. Neoplasia in zebrafish
(Danio rerio ) treated with N -methyl-N ¶-nitro-N -nitrosoguanidine by three
exposure routes at different developmental stages. Toxicol Pathol 2000;28:
716 – 25.
12. Haramis AP, Hurlstone A, van der Velden Y, et al. Adenomatous polyposis
coli-deficient zebrafish are susceptible to digestive tract neoplasia. EMBO Rep
2006;7:444 – 9.
13. Shepard JL, Amatruda JF, Finkelstein D, et al. A mutation in separase causes
genome instability and increased susceptibility to epithelial cancer. Genes Dev
2007;21:55 – 9.
14. Shepard JL, Amatruda JF, Stern HM, et al. A zebrafish bmyb mutation causes
genome instability and increased cancer susceptibility. Proc Natl Acad Sci U S A
2005;102:13194 – 9.
15. Beckwith LG, Moore JL, Tsao-Wu GS, Harshbarger JC, Cheng KC.
Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio ). Lab Invest 2000;
80:379 – 85.
16. Lee LM, Seftor EA, Bonde G, Cornell RA, Hendrix MJ. The fate of human
malignant melanoma cells transplanted into zebrafish embryos: assessment of
migration and cell division in the absence of tumor formation. Dev Dyn 2005;
233:1560 – 70.
17. Topczewska JM, Postovit LM, Margaryan NV, et al. Embryonic and
tumorigenic pathways converge via Nodal signaling: role in melanoma
aggressiveness. Nat Med 2006;12:925 – 32.
18. Lee JT, Herlyn M. Embryogenesis meets tumorigenesis. Nat Med 2006;12:
882 – 4.
19. Haldi M, Ton C, Seng WL, McGrath P. Human melanoma cells transplanted
into zebrafish proliferate, migrate, produce melanin, form masses and stimulate
angiogenesis in zebrafish. Angiogenesis 2006;9:139 – 51.
20. Nicoli S, Ribatti D, Cotelli F, Presta M. Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res 2007;67:
2927 – 31.
21. Stoletov K, Montel V, Lester RD, Gonias SL, Klemke R. High-resolution
imaging of the dynamic tumor cell vascular interface in transparent zebrafish.
Proc Natl Acad Sci U S A 2007;104:17406 – 11.
22. Mullins MC, Hammerschmidt M, Haffter P, Nusslein-Volhard C. Large-scale
mutagenesis in the zebrafish: in search of genes controlling development in a
vertebrate. Curr Biol 1994;4:189 – 202.
23. Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N.
Identification of 315 genes essential for early zebrafish development. Proc Natl
Acad Sci U S A 2004;101:12792 – 7.
30. Hurlstone AF, Haramis AP, Wienholds E, et al. The Wnt/h-catenin pathway
regulates cardiac valve formation. Nature 2003;425:633 – 7.
31. Liu TX, Zhou Y, Kanki JP, et al. Evolutionary conservation of zebrafish
linkage group 14 with frequently deleted regions of human chromosome 5 in
myeloid malignancies. Proc Natl Acad Sci U S A 2002;99:6136 – 41.
32. Kalev-Zylinska ML, Horsfield JA, Flores MV, et al. Runx1 is required for
zebrafish blood and vessel development and expression of a human RUNX1-2T1
transgene advances a model for studies of leukemogenesis. Development 2002;
129:2015 – 30.
33. Sabaawy HE, Azuma M, Embree LJ, Tsai HJ, Starost MF, Hickstein DD.
TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic
leukemia. Proc Natl Acad Sci U S A 2006;103:15166 – 71.
34. Onnebo SM, Condron MM, McPhee DO, Lieschke GJ, Ward AC.
Hematopoietic perturbation in zebrafish expressing a tel-jak2a fusion. Exp
Hematol 2005;33:182 – 8.
35. Langenau DM, Traver D, Ferrando AA, et al. Myc-induced T cell leukemia
in transgenic zebrafish. Science 2003;299:887 – 90.
36. Langenau DM, Feng H, Berghmans S, Kanki JP, Kutok JL, Look AT.
Cre/lox-regulated transgenic zebrafish model with conditional myc-induced
T cell acute lymphoblastic leukemia. Proc Natl Acad Sci U S A 2005;102:
6068 – 73.
37. Feng H, Langenau DM, Madge JA, et al. Heat-shock induction of T-cell
lymphoma/leukaemia in conditional Cre/lox-regulated transgenic zebrafish. Br J
Haematol 2007;138:169 – 75.
38. Langenau DM, Jette C, Berghmans S, et al. Suppression of apoptosis by bcl2 overexpression in lymphoid cells of transgenic zebrafish. Blood 2005;105:
3278 – 85.
39. Chen J, Jette C, Kanki JP, Aster JC, Look AT, Griffin JD. NOTCH1-induced
T-cell leukemia in transgenic zebrafish. Leukemia 2007;21:462 – 71.
40. Langenau DM, Keefe MD, Storer NY, et al. Effects of RAS on the genesis of
embryonal rhabdomyosarcoma. Genes Dev 2007;21:1382 – 95.
41. Le X, Langenau DM, Keefe MD, Kutok JL, Neuberg DS, Zon LI.
Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors
and hyperplasia in transgenic zebrafish. Proc Natl Acad Sci U S A 2007;104:
9410 – 5.
42. Patton EE, Widlund HR, Kutok JL, et al. BRAF mutations are sufficient to
promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr
Biol 2005;15:249 – 54.
43. Yang HW, Kutok JL, Lee NH, et al. Targeted expression of human MYCN
selectively causes pancreatic neuroendocrine tumors in transgenic zebrafish.
Cancer Res 2004;64:7256 – 62.
44. Amatruda JF, Shepard JL, Stern HM, Zon LI. Zebrafish as a cancer model
system. Cancer Cell 2002;1:229 – 31.
45. Oshima M, Oshima H, Kitagawa K, Kobayashi M, Itakura C, Taketo M.
Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal
polyps in mice carrying a truncated Apc gene. Proc Natl Acad Sci U S A 1995;92:
4482 – 6.
46. Di Cristofano A, Pesce B, Cordon-Cardo C, Pandolfi PP. Pten is essential for
embryonic development and tumour suppression. Nat Genet 1998;19:348 – 55.
47. Donehower LA, Harvey M, Slagle BL, et al. Mice deficient for p53 are
developmentally normal but susceptible to spontaneous tumours. Nature 1992;
356:215 – 21.
48. Wei K, Kucherlapati R, Edelmann W. Mouse models for human DNA
mismatch-repair gene defects. Trends Mol Med 2002;8:346 – 53.
49. Varley JM. Germline TP53 mutations and Li-Fraumeni syndrome. Hum
Mutat 2003;21:313 – 20.
24. Amsterdam A, Sadler KC, Lai K, et al. Many ribosomal protein genes are
cancer genes in zebrafish. PLoS Biol 2004;2:E139.
50. Galiatsatos P, Foulkes WD. Familial adenomatous polyposis. Am J
Gastroenterol 2006;101:385 – 98.
25. Moore JL, Rush LM, Breneman C, Mohideen MA, Cheng KC. Zebrafish
genomic instability mutants and cancer susceptibility. Genetics 2006;174:
585 – 600.
51. Bonneau D, Longy M. Mutations of the human PTEN gene. Hum Mutat
2000;16:109 – 22.
26. Wienholds E, Plasterk RH. Target-selected gene inactivation in zebrafish.
Methods Cell Biol 2004;77:69 – 90.
27. Berghmans S, Murphey RD, Wienholds E, et al. tp53 mutant zebrafish
develop malignant peripheral nerve sheath tumors. Proc Natl Acad Sci U S A
2005;102:407 – 12.
28. Faucherre A, Taylor GS, Overvoorde J, Dixon JE, Hertog JD. Zebrafish pten
genes have overlapping and non-redundant functions in tumorigenesis and
embryonic development. Oncogene 2008;27:1079 – 86.
29. Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in
cancer. Biochim Biophys Acta 2003;1653:1 – 24.
52. Chung DC, Rustgi AK. The hereditary nonpolyposis colorectal cancer syndrome: genetics and clinical implications. Ann Intern Med 2003;138:
560 – 70.
53. Furutani-Seiki M, Wittbrodt J. Medaka and zebrafish, an evolutionary twin
study. Mech Dev 2004;121:629 – 37.
54. Knudson AG. Hereditary cancer: two hits revisited. J Cancer Res Clin Oncol
1996;122:135 – 40.
55. Grabher C, Look AT. Fishing for cancer models. Nat Biotechnol 2006;24:
45 – 6.
56. Lam SH, Gong Z. Modeling liver cancer using zebrafish: a comparative
oncogenomics approach. Cell Cycle 2006;5:573 – 7.
Mol Cancer Res 2008;6(5). May 2008
Downloaded from mcr.aacrjournals.org on May 10, 2017. © 2008 American Association for Cancer Research.
693
694 Feitsma and Cuppen
57. Lam SH, Wu YL, Vega VB, et al. Conservation of gene expression signatures
between zebrafish and human liver tumors and tumor progression. Nat Biotechnol
2006;24:73 – 5.
62. Stern HM, Murphey RD, Shepard JL, et al. Small molecules that
delay S phase suppress a zebrafish bmyb mutant. Nat Chem Biol 2005;1:
366 – 70.
58. Spitsbergen J. Imaging neoplasia in zebrafish. Nat Methods 2007;4:
548 – 9.
63. Lally BE, Geiger GA, Kridel S, et al. Identification and biological evaluation
of a novel and potent small molecule radiation sensitizer via an unbiased screen of
a chemical library. Cancer Res 2007;67:8791 – 9.
59. Goessling W, North TE, Zon LI. Ultrasound biomicroscopy permits in vivo
characterization of zebrafish liver tumors. Nat Methods 2007;4:551 – 3.
64. Mallo M. Controlled gene activation and inactivation in the mouse. Front
Biosci 2006;11:313 – 27.
60. Daroczi B, Kari G, McAleer MF, Wolf JC, Rodeck U, Dicker AP. In vivo
radioprotection by the fullerene nanoparticle DF-1 as assessed in a zebrafish
model. Clin Cancer Res 2006;12:7086 – 91.
65. McGuire SE, Roman G, Davis RL. Gene expression systems in Drosophila :
a synthesis of time and space. Trends Genet 2004;20:384 – 91.
61. Sidi S, Look AT. Small molecules thwart crash and burn. Nat Chem Biol
2005;1:351 – 3.
66. Ellingsen S, Laplante MA, Konig M, et al. Large-scale enhancer detection in
the zebrafish genome. Development 2005;132:3799 – 811.
Mol Cancer Res 2008;6(5). May 2008
Downloaded from mcr.aacrjournals.org on May 10, 2017. © 2008 American Association for Cancer Research.
Zebrafish as a Cancer Model
Harma Feitsma and Edwin Cuppen
Mol Cancer Res 2008;6:685-694.
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